This cross-sectional study with 1,249 workers from all 49 municipal primary health care centers was conducted in Florianópolis, Santa Catarina State, Brazil, with the objective of investigating the prevalence of poor self-rated health and its association with working conditions and other factors. Multivariate statistical analyses were conducted using Poisson regression. The prevalence of poor self-rated health was 21.86% (95%CI: 19.56%-24.15%). The largest prevalence was found among dental assistants (35.71%), and the lowest among physicians (10.66%). In the adjusted analysis, the outcome was associated with female gender (PR = 1.48; 95%CI: 1.03-2.14), older age (PR = 1.29; 95%CI: 1.05-1.59), higher education (PR = 0.69; 95%CI: 0.55-0.87), more time working at the primary care center (PR = 1.57; 95%CI: 1.29-1.98), higher workload score (PR = 1.67; 95%CI: 1.35-2.05), obesity (PR = 1.74; 95%CI: 1.37-2.21), and often or always experiencing musculoskeletal symptoms (PR = 2.69; 95%CI: 1.90-3.83). A higher workload score remained associated with the outcome, suggesting an association between working conditions and self-rated health.